Citation of any document herein is not an admission that the document is prior art, or considered material to patentability of any claim herein, and any statement regarding the content or date of any document is based on the information available to the application at the time of filing and does not constitute an affirmation or admission that the statement is correct.
Healthy animals or animals that are not infected by pathogenic organisms, such as pathogenic bacteria, grow faster and gain more weight per kilogram of feed. As a result, antimicrobial compounds have been used as growth promoters in farm animals since the 1940's. Typically, the antimicrobial compounds are administered in feed at a subtherapeutic or low dose.
While subtherapeutic doses of antimicrobial compounds have long been used to help farm animals maintain health and grow faster, recent reports demonstrate a link between the use of antibiotics and the presence of drug-resistant bacteria on the meat produced from these animals. As a result the European Commission, U.S. Department of Agriculture (USDA) as well as U.S. Food and Drug Administration (FDA) have all instituted bans and guidelines on the use of certain antibiotics as growth promoters. Currently, the regulations typically focus on the use of antibiotics that are the same as or similar to antibiotics used to treat humans. However, there is growing opposition in general to the use of all antibiotic drugs to enhance the growth of farm animals. Furthermore, the market for organically raised meat is increasing and to be certified organic, U.S. meat must come from animals raised without antibiotics. As a result there is a need for a new growth enhancer for farm animal feed.
In addition, companion animal health is a fast-growing market. Maintaining the health of companion animals (e.g., aging companion animals) via stimulation of innate immunity is an attractive approach to health maintenance in companion animals, and an orally delivered immune stimulant would be highly valued.
Brevibacillus texasporus (BT) (e.g., ATCC PTA-5854) is a recently identified soil bacterium that produces a group of cationic NRPS peptides (see, WO 2005/074626, Wu et al. 2005, and GenBank Accession No. AY953371). The cationic peptides from BT display a broad-spectrum of antibacterial activity in vitro, killing gram positive and negative bacteria, fungi and protozoa (WO 2005/074626). However, the high degree of 16S rDNA sequence identity (98.5%) between PTA-5854 and Brevibacillus laterosporus, a species defined almost exclusively by strains lacking the BT peptides, requires classification of Brevibacillus texasporus as a subspecies of Brevibacillus laterosporus (i.e., Brevibacillus laterosporus subsp. texasporus). Therefore, “Brevibacillus texasporus”, “Brevibacillus laterosporus subsp. texasporus” and “B. texasporus” are synonymous and are used interchangeably when describing strains of the BT-peptide-producing subspecies.
Despite the in vitro antibacterial activity, the BT peptides seem to lack antibacterial activity in vivo. Vancomycin-resistant enterococci are highly sensitive to the BT peptides in vitro. However, the BT peptides at concentrations well above the minimal inhibition concentrations fail to decolonize commensal VRE from the mouse GI track.
However, an isolated peptide was shown to be effective in preventing colibacillosis and salmonellosis in chickens when used as a feed additive. In addition, this isolated peptide was also shown to be effective in promoting growth and increasing feed conversion in chickens.
Perhaps more importantly, the in vivo effect of the BT peptides appears to be independent of its in vitro antibiotic activities, as it is effective in preventing infections in chickens by E. coli and Salmonella at concentrations below its in vitro minimal inhibition concentrations (JIANG et al. 2005; KOGUT et al. 2007; KOGUT et al. 2010). It is also noted that blood heterophils and monocytes are primed for activation in chickens fed the BT peptides, pointing to innate immunostimulation as a likely mode of action. These features make the BT peptides an ideal feed additive and alternative to antibiotic compounds in farm animal production.
In addition, since innate immunity is now also known to play key roles in controlling viral and fungal infections as well as in preventing non-infectious diseases such as obesity/Metabolic Syndrome and type 2 diabetes mellitus (VIJAY-KUMAR et al. 2010), the BT innate immunity modulator should also have important applications in these therapeutic areas.
However, the economical value of the BT peptides as a feed additive is severely limited by the need to isolate the peptides, which increases the cost of production to a point where it is no longer economically viable. As an alternative to purifying the peptides, it is possible that the entire organism (PTA-5854), as discussed in international patent publication WO 2005/074626, could be used directly for the production of a feed additive.
Typically, a direct-fed microbial (DFM) or probiotic strain needs to reach the intestine in a viable form and in sufficient numbers, which requires the survival of the strain during feed processing and digestion (see, U.S. Pat. No. 5,480,641 and U.S. Patent Publication 20040247568). Since feed pellet production typically involves enough heat to significantly reduce viability, most probiotic strains are selected for their resistance to heat and the pH conditions found in the stomach. Currently the most stable probiotic strains are Bacillus spores, since bacterial spores are heat resistant and stay viable during long-term storage.
However, since the BT organism is not believed to be a symbiotic bacterium normally found in the intestinal track of farm animals, it is desirable and/or necessary to inactivate the organism before use. However, in spore producing strains this is problematic. In addition, at least one governmental regulatory authority considers the following criteria when assessing novel feeds involving microbial sources: safety of the production of the microorganism; safety of the microbial product to humans, animals and the environment; potential impact of horizontal gene transfer; interactions with gastrointestinal microflora; persistence in the gut; potential impact on humans and the environment due to shedding of viable microorganisms, particularly if there are perceived health impacts due to contamination of the meat (Directive on Guidelines for the Assessment of Novel Feeds: Microbial Sources, Draft—June 2007, The Canadian Food Inspection Agency). Therefore, when using microorganisms as a food additive there are two conflicting desired outcomes. The first is to maintain the organism in a viable condition throughout processing and digestion and the second diametrically opposed desire is to completely or nearly completely inactivate the organism without inactivation of the active peptides. An additional benefit of inactivation is that it removes or reduces the chances of horizontal gene transfer between B. texasporus and microorganisms in the gut and in the environment; eliminates or limits potential interactions with the gastrointestinal (GI) microflora; eliminates or reduces the potential risk on humans and the environment through shedding of viable cells; and/or eliminates or reduces contamination of the meat derived from the animal consuming the feed. Therefore, continuous spore formation by PTA-5854 severely limits the ability to use this strain as a DFM, since the spores are extremely resistant to most methods used to inactivate vegetative cells, mandating harsh and expensive methods for their removal.
As a result, there is a need in the art for a strain of Brevibacillus texasporus that can be used effectively as an inactivated DFM.